Efforts aimed at stimulating immune responses by modifying human immunodeficiency virus (HIV) antigens and using various delivery systems and adjuvants have so far failed to produce a HIV vaccine that protects against primary infections, highlighting the need for the continued development of alternative and effective vaccine candidates. A common feature of many of these approaches is the use of positive immunostimulation to enhance the immune response. Less often considered in the context of vaccine design however are the inhibitory signals that serve to moderate the immune response. Antigen-presenting cells, such as dendritic cells (DC), play a critical role in the establishment and maintenance of the immune responses against HIV infection;therefore DCs are key targets for vaccines that stimulate immune signaling. Our proposal is based on findings that the suppressor of cytokine signaling 1 (SOCS1) protein functions as an antigen-presentation attenuator by restricting the Janus-activated kinase-signal transducers and activators of transcription and Toll-like receptor-signaling pathways. Inhibition of SOCS1 signaling in DCs was shown to induce higher levels of both Th1- and Th2-polarizing cytokines, resulting in the enhancement of memory, antigen-specific T- and B- cell responses and activated natural killer cells. Herein we hypothesize that the immunogenicity of an adenovirus (Ad)-based HIV vaccine will be enhanced by suppressing the SOCS1 signaling pathway. To test this hypothesis, the Specific Aims of the project are to (1) inhibit DC SOCS1 signaling with Ad-mediated delivery of SOCS1 small interfering (si) RNA, (2) evaluate the effect(s) of SOCS1-silencing on DC function, and (3) determine the effect of SOCS1-silencing in Ad-targeted DCs by measuring the vaccination efficiency of HIV antigen-specific immunes responses. To accomplish Aim 1 we will first generate Ad vectors that express SOCS1 siRNA. Then, using quantitative RT-PCR and Western blotting methods, we will show that DCs infected with the siRNA-expressing Ad produce significantly reduced levels of SOCS1 mRNA and protein. In the second aim we will determine the effects of SOCS1-silencing on DC function. This will be done by flow cytometric analyses of DC phenotypic markers and cytokines expression profiles. The third phase of the project is designed to quantitatively assess the immune responses to an HIV vaccine in animals with transiently SOCS1-silenced DCs. This aim will be accomplished by measuring HIV-specific humoral and cellular-mediated responses and the ability of the vaccination protocol to mitigate an in vivo viral challenge. The negative regulation of signal transduction pathways exerted by SOCS1 is vitally important for an appropriate response to cytokine stimulation, however by transiently tipping the balance this project represents a potentially novel direction in the design of HIV vaccines. By "inhibiting the inhibitors" of antigen-presentation, such a SOCS1, this project represents a generally applicable and alternative strategy for enhancing the potency of prophylactic and therapeutic HIV vaccines. PUBLIC HEALTH RELEVANCE: Over 40 million people are currently infected with HIV and, considering the lack of a prophylactic vaccine in the foreseeable future, this number is expected to rise exponentially particularly in the underdeveloped regions of the world. The goal of this project is to evaluate whether the efficacy of an HIV vaccine can be significantly improved by modulating key effectors of the immune response. The relevance of such an improvement lies in the development and potential global impact of a vaccine that affords protection from HIV infection.